Apoptosis plays critical roles in many diseases such as stroke, neurodegenerative diseases, autoimmune diseases and cancer. In the cells undergoing apoptosis, the multi-domain proapoptotic BCL-2 family proteins such as BAX or BAK are activated to damage the mitochondrial membrane by forming large pores. This results in the release of protein factors into the cytoplasm from the mitochondrial intermembrane space to amplify the cascade events of apoptosis in the cytoplasm. The goal of this research is to understand the mechanism of the pore formation by BAX or BAK in the mitochondria. The hypothesis is that in apoptotic cells the proapoptotic BCL-2 proteins BAX and BAK form domain-swapped dimers and these domain-swapped dimers further oligomerize via reciprocal exchange of their BH3 domains, leading to the permeabilization of the mitochondrial outer membrane. The specific aims of this research are 1) to investigate the interface formed by the BH3 (BCL-2 homology domain 3) domains in the oligomeric BAK pore by the site-directed spin labeling (SDSL) method of electron paramagnetic resonance (EPR) spectroscopy, 2) to test the existence of the domain-swapped dimer of BAK in the BAK oligomeric pore by the SDSL method of EPR, and 3) to develop a method to determine the orientation of the helices in the channel-forming domain (helices 5-6) relative to the membrane normal using an oriented membrane and the angular dependence of the dipolar line broadening by the SDSL methodology. For the first two aims, the distance between the residues proposed to exist in the protein-protein contact interfaces will be measured by the continuous wave (CW) method or by the double electron resonance (DEER) method. Additionally, the conformational changes in BAK associated with the dimerization and oligomerization processes will be determined by the solvent accessibility measurements using the power saturation method. The depths of the spin-labeled residues in the membrane will also be measured by the same method. For the third aim, a doubly spin labeled -helix will be prepared in oriented membranes. The dependence of the EPR signal on the relative orientation of the aligned sample to the magnetic field will be measured by recording the EPR spectra at various angles between the magnetic field and the membrane normal. A theoretical method will be developed to explain the angular dependence of the EPR signal. This approach will provide a novel means to determine the orientation of other membrane- inserted helices (including those of BAX or BAK) relative to the membrane normal. The outcome of this research will provide detailed understanding of the molecular mechanism of mitochondrial permeabilization by BAK and BAX. This in turn will provide valuable insights in designing therapeutic means to control the cell death processes either by enhancing apoptosis in cancer or by suppressing it in strokes or neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Apoptosis plays critical roles in many diseases such as stroke, neurodegenerative diseases, autoimmune diseases and cancer. Mitochondrial permeabilization by BAX or BAK constitutes a major control point in the mitochondrial programmed cell death or apoptosis pathways. The goal of this research is to understand the mechanism of pore formation by BAX or BAK in the mitochondria, which would provide new insights as to how to control the programmed cell death in diseases.